1. Field of the Invention
The present invention relates generally to the fields of molecular biology and cellular biology of cytokines. More specifically, the present invention relates to a methods of inhibiting or enhancing the TGF-xcex2-Smad signaling pathway.
2. Description of the Related Art
The TGF-xcex2 family of polypeptide growth factors regulate cell division, differentiation, motility, adhesion and death in virtually all metazoan tissues39,44,46,51,53,56. Members of this family include the TGF-xcex2s, the activins, the bone morphogenetic proteins (BMPs) and other related factors. Signal transduction by these factors involves three classes of molecules: a family of membrane receptor serine/threonine kinases, a family of cytoplasmic proteins, the Smad family, that serve as substrates for these receptors, and nuclear DNA-binding factors that associate with Smads forming transcriptional complexes43,52. Signaling is initiated by binding of the growth factor to a specific pair of receptor kinases, an event that induces the phosphorylation and activation of one kinase, known as the xe2x80x9ctype I receptorxe2x80x9d, by the other kinase or xe2x80x9ctype II receptorxe2x80x9d65. The activated type I receptor phosphorylates a subset of Smads, known as xe2x80x9creceptor-regulated Smadsxe2x80x9d (R-Smads), which then move into the nucleus43,52. On their way to the nucleus, R-Smads associate with the related protein Smad49, a tumor suppressor gene product1. In the nucleus, this complex may associate with specific DNA-binding proteins that direct it to the regulatory region of target genes. The first identified Smad-associated DNA-binding factor was the forkhead family member Fast1, which mediates activation of Mix.2 in response to activin-type signals during Xenopus embryogenesis36. The integrity of this signaling network is essential for normal development and tissue homeostasis, and its disruption by mutation underlies several human inherited disorders and cancer43,52.
Because of the diversity of processes controlled by different TGF-xcex2 family members, there is an intense interest in elucidating the basis for the specificity of their signal transduction pathways. The TGF-xcex2 and activin type I receptors, which have nearly identical kinase domains31,60, interact with and phosphorylate Smad2 (or the closely related Smad3)16,40,30,54,8 which then interacts with DNA-binding factors such as Fast134,33,49. The BMP receptors interact with Smad1 (or the closely related Smads 5, 8 or, in Drosophila, Mad)35,40,11,14,18,10 which do not recognize Fast136. Although the TGF-xcex2 and BMP pathways are well segregated from each other, their receptors and R-Smads are structurally very similar. The specificity of the receptor and Smad interactions in each pathway may therefore be dictated by discrete structural elements.
The Smad4/DPC4 tumor suppressor1 is inactivated in nearly one half of pancreatic carcinomas2 and to a lesser extent in a other cancers2-4. Smad4/DPC4, and the related tumor suppressor Smad2, belong to the Smad family of proteins which mediate TGFxcex2/activin/bone morphogenetic protein (BMP)-2/4 cytokine superfamily signaling from the receptor serine/threonine protein kinases at the cell surface to the nucleus5-7. Smad proteins, which get phosphorylated by the activated receptor, propagate the signal, in part, through homo-oligomeric and hetero-oligomeric interactions8,3. Smad4/DPC4 plays a central role as it is the shared hetero-oligomerization partner of the other Smads. The conserved C-terminal domains of Smads are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation.
The conserved C-terminal domain of Smad family members is the likely effector domain, whereas the conserved N-terminal domain is the likely negative regulator of activity14. When overexpressed in a Smad4/DPC4xe2x88x92/xe2x88x92 cell line, the Smad4/DPC4 C-terminal domain activates the transcription of TGF-xcex2 responsive genes and results in growth arrest in a ligand-independent manner, paralleling the effects of the TGF-xcex2 ligand9. In addition, microinjection of mRNAs encoding the C-terminal domain of Smad2 into Xenopus embryos can induce a mesoderm response that mimics the effects of the full-length protein16. Furthermore, the Smad4/DPC4-C-terminal domain fused to a heterologous DNA-binding domain can activate gene expression from a reporter construct14. Consistent with the Smad C-terminal domain being the main effector domain, the majority (10 out of 13) of the tumorigenic missense mutations in Smad4/DPC4 and Smad2, as well as mutations isolated from Drosophila and C. elegans genetic screens map to the C-terminal domain.
The prior art is deficient in the lack of effective means of inhibiting or enhancing the TGF-xcex2-Smad signaling pathway. The present invention fulfills this longstanding need and desire in the art.
It is an object of the present invention to use the L3 loop of the Smad proteins 1, 2, 3, 4, 5 or 6 or the C-terminal tail of Smad proteins 1, 2, 3, 4 or 5 in protein-interaction assays to screen for agents that increase or decrease Smad interactions via these regions.
It is another object of the present invention to provide a method of screening for drugs that interfere with or enhance signaling by TGF-xcex2 or other members of the TGF-xcex2 family that signal through Smad proteins.
It is another object of the present invention to provide a screening method that utilizes high specificity peptide-Smad interactions and peptide receptor interactions and is suitable for adaptation to high throughput assays.
In one embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad binding to a complementary Smad via the L3 loop region, comprising the steps of: a) producing a synthetic Smad polypeptide encompassing the L3 loop region; b) attaching a detectable label onto this polypeptide; c) contacting the synthetic L3 loop polypeptide with a complementary Smad protein immobilized on a solid support; d) measuring the amount of labeled L3 loop polypeptide bound; e) in parallel to steps (c) and (d), conducting these same steps in the presence of a test substance; and f) comparing the amount of L3 loop polypeptide bound in the presence of a test substance with the amount bound in the absence of test substance so as to identify test substances that either increase L3 loop polypeptide binding to the Smad protein or decrease L3 loop polypeptide binding to the Smad protein.
In another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad binding to a complementary Smad via the L3 loop region, comprising the steps of: a) producing a synthetic Smad polypeptide, encompassing the L3 loop region as defined by the crystal structure of the Smad4/DPC4 C-terminal domain; b) producing this polypeptide containing a chemical group that allows immobilization; c) contacting this L3 loop polypeptide with a labeled complementary Smad protein; d) measuring the amount of labeled Smad protein bound to the L3 loop polypeptide; e) in parallel to steps (c) and (d), conducting these same steps in the presence of a test substance; and f) comparing the amount of Smad protein bound in the presence of a test substance with the amount bound in the absence of test substance in order to identify test substances that either increase L3 loop polypeptide binding to the Smad protein or decrease L3 loop polypeptide binding to the Smad protein.
In yet another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad4 binding to a complementary Smad via the C-terminal phosphorylated tail (xe2x80x9cC-tailxe2x80x9d) of this Smad, comprising the steps of: a) producing a synthetic polypeptide corresponding to the C-terminal tail of a given Smad encompassing the C-terminal tail that follows the H5 alpha-helix as defined by the crystal structure of the Smad4/DPC-terminal domain; b) attaching a detectable label onto this polypeptide; c) contacting this C-tail polypeptide with Smad4 protein immobilized on a solid support; d) measuring the amount of labeled C-tail polypeptide that is bound to Smad4; e) in parallel to steps (c) and (d), conducting these same steps in the presence of a test substance; and f) comparing the amount of C-tail bound in the presence of a test substance with the amount bound in the absence of the substance in order to identify test substances that either increase C-tail polypeptide binding to the Smad protein or decrease C-tail polypeptide binding to the Smad protein.
In yet another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad4 binding to a complementary Smad via the C-terminal phosphorylated tail (xe2x80x9cC-tailxe2x80x9d) of this Smad, comprising the steps of: a) producing a synthetic polypeptide corresponding to the C-terminal tail of a given Smad encompassing the C-terminal tail that follows the H5 alpha-helix as defined by the crystal structure of the Smad4/DPC4 C-terminal domain; b) producing this polypeptide containing a chemical group that allows immobilization; c) contacting this derivative C-tail polypeptide with the labeled Smad4 protein; d) measuring the amount of labeled Smad4 bound to the C-tail polypeptide; e) in parallel to steps (c) and (d), conducting these same steps, in the presence of a test substance; f) comparing the amount of Smad4 bound in the presence of a test substance, with the amount bound in the absence of test substance in order to identify test substances that either increase Smad4 binding to the C-tail polypeptide or decrease Smad4 binding to the C-tail polypeptide.
In yet another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit Smad binding to a receptor of the TGF-xcex2 family, comprising the steps of: a) producing a synthetic polypeptide corresponding to the amino acid sequence of a given Smad encompassing the L3 loop region as defined by the crystal structure of the Smad4/DPC4 C-terminal domain; b) attaching a detectable label onto this polypeptide; c) contacting this L3 loop polypeptide with a receptor cytoplasmic domain protein such as a Smad1-derived L3 loop polypeptide with the bone morphogenetic protein receptor cytoplasmic domain, or contacting a Smad2-derived L3 loop polypeptide with the TGF-xcex2 receptor cytoplasmic domain) immobilized on a solid support; d) measuring the amount of labeled L3 loop polypeptide; e) in parallel to steps (c) and (d), conducting these same steps, in the presence of a test substance; and f) comparing the amount of L3 loop polypeptide bound in the presence of a test substance with the amount bound in the absence of test substance in order to identify test substances that either increase L3 loop polypeptide binding to the receptor or decrease L3 loop polypeptide binding to the receptor.
In yet another embodiment of the present invention, there is provided a method of screening for drugs which enhance or inhibit binding of a Smad N-terminal domain to the C-terminal domain of the same Smad protein, comprising the steps of: a) producing recombinant forms of the N-terminal domain and C-terminal domain polypeptides, with one containing a detectable label and the other containing a moiety allowing immobilization onto a solid support; b) contacting the recombinant N-terminal domain polypeptide with the C-terminal domain polypeptide; c) measuring the amount of labeled domain polypeptide bound; d) in parallel to steps (b) and (c), conducting these same steps, in the presence of a test substance; e) comparing the amount of labeled polypeptide bound in the presence of a test substance with the amount bound in the absence of a test substance so as to identify test substances that either increase N-terminal domain binding to the C-terminal domain or decrease N-terminal domain binding to the C-terminal domain.
Smad2 and Smad4 are related tumor suppressors that, in response to TGF-xcex2, form a complex that mediates transcriptional and growth inhibitory responses. The effector function of Smad2 and Smad4 is located in their conserved C-terminal domain (C domain) and inhibited by the presence of their N-terminal domains (N domain). The inhibitory function of the N domain is shown herein to involve a physical interaction with the C domain, preventing the association of Smad2 with Smad4. This inhibitory function is increased in tumor derived forms of Smad2 and 4 that carry a missense mutation in a conserved N domain arginine. The mutant N domains have increased affinity for their respective C domains, inhibit Smad2-Smad4 interaction and prevent TGF-xcex2-induced Smad2-Smad4 association and signaling. Whereas mutations in the C domain disrupt the effector function of the Smads, the N domain arginine mutations inhibit Smad signaling through a gain of autoinhibitory function. Gain of autoinhibitory function provides a novel mechanism of tumor suppressor inactivation.
In the present invention, the crystal structure of the C-terminal domain (CTD) of the Smad4/DPC4 tumor suppressor, was determined at 2.5 xc3x85 resolution and revealed that the Smad4/DPC4-C-terminal domain forms a crystallographic trimer through a conserved protein-protein interface to which the majority of the tumor-derived missense mutations map. These mutations disrupt homo-oligomerization in vitro and in vivo, suggesting that the trimeric assembly of the Smad4/DPC4 C-terminal domain is a critical function in signaling that is targeted by tumorigenic mutations.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.